Stem cells show differential characteristics as they are able to sustain themselves and differentiate into one or more cell type. Although research into stem cells and their applications is still in its early stages, adult stem cells in bone marrow have been used in transplants for more than 30 years. Nevertheless, in recent years, stem cell technology has made large advances such that stem cells are currently considered as a promising source of tissue and organs, with an important therapeutic potential for repair and regeneration of tissues.
The use of stem cells is an alternative therapy for several human diseases, particularly those in which there is a loss of functional cells, including chondral, bone and muscular lesions, neurodegenerative diseases, immunologic rejection, heart disease and skin disorders (see U.S. Pat. Nos. 5,811,094, 5,958,767, 6,328,960, 6,379,953, 6,497,875).
In addition to cell therapy applications, stem cells have potential applications in the research and development of new drugs. On the one hand, the study of mechanisms implicated in the proliferation and differentiation of stem cells is of great value in the process of searching for and characterizing new genes involved in a wide range of biological processes, including cell development and differentiation and neoplastic processes (Phillips et al., 2000; Ramalho-Santos et al., 2002; Ivanova et al., 2002). On the other hand, stem cell technology allows specialized cells to be generated and the development of cell models for human and animal diseases, in which the efficacy and toxicity of new active ingredients can be determined in the preclinical phase (U.S. Pat. No. 6,294,346).
An adult somatic stem cell is an undifferentiated cell which is found in differentiated tissue and which has the capacity to proliferate and differentiate into one or more cell types. Adult stem cells are present in different adult tissue, their presence being extensively reported in bone marrow, blood, cornea, retina, brain, muscle, skeleton, dental pulp, gastrointestinal epithelium, liver and skin (Jiang et al., 2002). By their nature, adult stem cells can be used in an autologous setting, and as such, they are immunologically compatibles and their use does not raise any ethical concerns.
An adult stem cell should be able to give rise to fully differentiated cells with mature phenotypes which are integrated into the tissue where they are found and which are able to carry out the specialized functions of the given tissue. The term “phenotype” refers to observable characteristics of the cell, such as characteristic morphology, interactions with other cells and with the extracellular matrix, cell surface proteins (surface markers) and characteristic functions.
Different populations of adult stem cells capable to contribute to the repair of different tissues have been described. Among these populations, those of mesodermic origin are of particular interest because they offer the theoretical possibility of regenerating a large number of clinically very relevant connective tissues such as bone, cartilage, tendons, skeletal muscle, heart muscle, vascular endothelium, sub-dermal fat and bone marrow stroma. The first cell population of this type isolated was the so-called mesenchymal stem cells (MSC), which are found in bone marrow stroma (Friedenstein et al., 1976; Caplan et al., 1991; Pittenger et al., 1999). These cells have been extensively characterized and studies performed with these cells have shown that they can differentiate into different mesenchymal cell lines such as adipocytes (Beresford et al., 1992), chondrocytes (Johnstone et al., 1998), myoblasts (Wakitani et al., 1995) and osteoblasts (Haynesworth et al., 1992). Likewise, they also have the capacity to differentiate into neurons (Sanchez-Ramos et al., 2000).
The ideal source of adult stem cells is one in which they can be obtained by an easy, non-invasive process and one that allows a sufficient number of cells to be isolated. In particular, a source should provide stem cells that can be easily isolated from a living subject without significant risks and discomfort and the source should allow a high yield to be obtained with minimal contamination from other cell types, without excessive cost of isolation and culture.
The process of obtaining bone marrow is painful and the yield is very low, a substantial increase in the number of cells being necessary by ex vivo expansion, to obtain clinically relevant amount. This step increases cost and makes the procedure time consuming, as well as increases the risk of contamination and loss of material. For these reasons, it would be very desirable to be able to isolate multipotent cells from mesenchymal tissues other than bone marrow. In particular, given their surgical accessibility, it would be convenient to be able to isolate cells from non-osteochondral mesodermal tissues such as, but not limited to, skin, fat and muscle tissue.
The presence of different populations of multipotent adult cells in soft tissues derived from the embryonic mesoderm has been reported by several authors. For example, it has been reported that multipotent cells can be obtained from skeletal muscle and other connective tissue of mammals (Young et al. 1993, Rogers et al. 1995). Multipotent cells have also been obtained from human lipoaspirated tissue (Zuk et al., 2001). Another example of multipotent cells isolated from adult connective tissue is the so-called Multipotent Adult Progenitor Cells (MAPC) obtained from bone marrow (Jiang et al., 2002). In principle, all these isolated cell populations could be used in the repair and regeneration of connective tissue in a similar fashion to the MSC of bone marrow (Caplan et al., 2001). However, except for MAPC, none of these populations has been, until present, sufficiently characterized at the phenotype level. Therefore, although the presence of multipotent adult cells has been described in different connective tissues, in the current state of the art, it is not possible to identify and unequivocally distinguish between different multipotent cell types obtained from soft tissue, or to obtain a substantially pure population.
Currently, phenotype characterization of stem cells comprises determination of markers such as cell surface receptors, among others; and the determination of their capacity for differentiation in vitro cultures. Each cell type has a certain combination of surface markers, that is, it has a certain profile of expression that characterizes that particular cell type, distinguishing it from others.
Different combinations of surface markers have been used for identifying and isolating substantially pure populations of hematopoietic stem cells from the bone marrow of mice, such as: [Linneg/low, Thy1.1low, c-Kithigh, Sca-1+j, [Lin−, Thy1.1low, Sca-1+, rhodamine 123low](Morrison, S. J. et al., 1995) or [Lin−, CD34−/int, c-Kit+, Sca-1+] (Osawa, M. et al., 1996). Likewise, similar combinations of markers have been used for enriching populations of human hematopoietic stem cells [Lin−, Thy1+, CD34+, CD38neg/low](Morrison, S. J. et al., 1995).
Currently, it is not known how many markers associated with compromised and differentiated cells are also present in the different multipotent adult mesenchymal cell populations. For example, a commonly used marker for enriching multipotent adult mesenchymal cells is CD44 (hyaluronic acid receptor). Nevertheless, CD44 is also present in different types of compromised and differentiated cell types. The uncertainty about which markers are associated with the stem cells to allow them to be distinguished from those cells that show a greater degree of differentiation, along with the low percentage of stem cells present in adult cells, has made it difficult to identify and purify populations of multipotent adult mesenchymal cells.
A significant disadvantage in using multipotent adult cells resides in the fact that most of the current sources for obtaining multipotent adult cells are contaminated with other cell types, complicating the process of identification, isolation and characterization of the populations of multipotent adult cells with the objective of using them for therapeutic or other ends. Thus, there is an interest in obtaining a population of multipotent adult cells isolated in a substantially pure form.
The characterization of a multipotent adult cell population from non-osteochondral mesenchymal tissue will allow a method for identification and isolation to be designed, as well as the identification of growth factors associated with self-regeneration. Moreover, there may be growth factors associated with the initial phases of differentiation, knowledge of which would allow more efficient in vivo and ex vivo differentiation, as well as for exercising control over the proliferation of stem cells.
The present invention provides a multipotent adult cell population from non-osteochondral mesenchymal tissue, preferable from adipose tissue, isolated and characterized by means of immunophenotype markers present on the cell surface, showing their multipotent nature.
Similarly, the present invention provides a method for the identification and isolation of a population of multipotent adult cells from non-osteochondral mesenchymal tissue, dependent on a pattern of characteristic immunophenotype markers, allowing a composition of substantially homogeneous multipotent stem cell markers to be obtained.